Hypercholesterolemia is an important risk factor definitively connected with cardiovascular disease and, particularly, with atherosclerosis and coronary heart disease. Millions people in the world suffer from coronary heart disease, which is the leading cause of death and morbidity in a productive age, especially in Western Europe and in the United States. For this reason it is also a significant drain on healthcare resources in the western world. For example, in the USA total costs (direct and indirect) connected with the disease were estimated as about $118 billion in 2000 for 1.1 million citizens experienced myocardial infarction, more than 40% of those died [Terry A. Jacobson, Clinical Context: Current Concepts of Coronary Heart Disease Management, Am J Med. 2001; 110 (6A):3S11S].
Cholesterol is the principal sterol of humans and higher animals. It is an important component of cell membranes and lipoproteins, and a key biosynthetic precursor of bile acids and steroidal hormones. It is found in all body tissues and also among the lipids in the bloodstream, but with especially high content in the brain, spinal cord and in animal fats. Nevertheless, an increased level of cholesterol in blood, especially in its low-density lipoprotein-bound form, is the critical factor in the development of coronary heart disease, which creates a danger of heart attack.
Because of insolubility of cholesterol, it is transported in the blood in a modified form of lipoproteins. There are a number of lipoproteins differing in their properties and physiological role, but the most important are low-density lipoprotein (LDL) and high-density lipoprotein (HDL).
LDL is a major carrier of cholesterol in the blood and, as mentioned above, is the major risk factor in the development of coronary heart disease. Its excess in the blood leads to a situation when arteries are blocked to a greater or lesser extent by the deposition of cholesterol plaques that means a condition of stenosis or atherosclerosis in particular. This is the reason of thrombus formation blocking blood flow in coronary or cerebral arterial vessels.
When the flow is directed to a part of the heart muscle, this cause a heart attack. If a thrombus blocks the blood flow to a certain region of the brain, the consequence is a stroke.
In contrast to LDL, HDL carries away to the liver the excess of cholesterol from tissues. It is considered to be able to remove some cholesterol from atherosclerotic plaques thus making slower their growth. Angiographical studies showed a correlation between elevated levels of HDL and a decreased number of sites of stenosis in the coronary arteries of humans. This means a protective action of HDL against heart attack and indicates a possibility to use measuring the level for a prognosis of higher or lower risk.
Demand of the body in cholesterol is covered by two sources: by the biosynthesis that mainly proceeds in the liver, intestine and skin, and via taking up from food, mainly from animal and dairy products. Under mixed diet, ratio of cholesterol amounts supplied by the sources is about 1:1.
Cellular cholesterol homeostasis is very important for the prevention of coronary heart disease. In general, the plasma concentration of cholesterol in the body is regulated by the dietary cholesterol absorption, by the biosynthesis of cholesterol itself and its esterified forms, by the metabolic removal of circulating cholesterol, and by the excretion of cholesterol via bile and feces.
Both diet and genetically determined biosynthetic-metabolic specificity of the body are instrumental in the development of atherosclerosis. A diet high in cholesterol will lead to a high level of cholesterol in the bloodstream that has important consequences. Dietary cholesterol suppresses the biosynthesis of cholesterol in the body, especially in tissues other than the liver. A parallel effect is inhibition of synthesis of LDL receptors. As a result of reduction in the number of receptors, the level of LDL in blood increases, leading to the deposition of atherosclerotic plaques. Damaged biosynthesis of LDL receptors could be also a result of a genetic deviation. A good regulation of cholesterol biosynthesis is very important. Oxygenated derivatives of cholesterol seem to control the biosynthesis of the responsible enzymes in a receptor-mediated process, providing in this way feedback regulation for the biosynthesis of cholesterol. Thus, modern approaches to prevention of atherosclerosis are based on the correction of both external and internal factors ruling the cholesterol level in blood: dietary supply and absorption of exogenic cholesterol, on the one hand, and the biosynthesis of endogenic cholesterol and related structures, on the other hand.
The first approach is realized via diet modification, such as reduction of dietary supply of cholesterol, for example by partial substitution of food animal fats by plant fats that do not contain cholesterol. The reduction of dietary cholesterol absorption can be reached via application of special food additives or foods enriched by the abundant phytosterols, such as beta-sitosterol or campesterol, or their saturated derivatives (stanols). Plant sterols produce anticholesterolemic effect which is considered to be connected with the inhibition of cholesterol absorption in the intestine because of competition with cholesterol for incorporation into micelles, although other absorption steps may also be involved. When the plant sterols replace cholesterol of the micelles, free cholesterol is excreted with feces. A limitation of the approach is that relatively large doses of sterols are required for modest reduction in plasma cholesterol. The same is true in respect to other agents blocking cholesterol absorption, such as stanols, aminoglycoside antibiotic neomycin, which appears to inhibit cholesterol absorption by forming complexes with cholesterol that are excreted, and the bile salt binder cholestyramine, an anion exchanger that indirectly alters cholesterol levels by limiting the resorption of cholesterol-derived bile salts.
In realization of the second approach, inhibitors of cholesterol (and its derivatives) biosynthesis play an important role and the search for new agents with this activity now constitutes the major research efforts. For a number of years much research went into the development of competitive inhibitors for 3-hydrohy-3-methylglutaryl coenzyme A reductase, a major regulatory enzyme of cholesterol biosynthesis. Many attempts to use for this purpose oxygenated sterols, which via binding oxysterol receptors were expected to decrease activity of HMG-CoA reductase, did not bring practical results. A breakthrough came with the discovery of a series of fungal metabolites with very high affinities for the reductase found to be highly efficient inhibitors of cholesterol biosynthesis. Nowadays these compounds and some synthetic analogs, commonly known as statins, are available commercially and widely used.
Although being relatively safe and efficient in treatment and prevention of coronary heart disease, statins have certain limitations in their use and they need care in application because of possible side effects. Thus, patients taking them respond very often to the lowering of cholesterol biosynthesis by a compensatory enhancement of cholesterol absorption from food and, especially for the cases when statins are used as a monotherapy, failed to reach treatment goals. Risk of liver complications, dictates the use of statins under medical control. Taking additionally into account relatively high costs of the therapy, which vary from $20,000 to $40,000 per quality-adjusted life-year saved [John A. Farmer, Economic Implications of Lipid-Lowering Trials: Current Considerations in Selecting a Statin, Am J Cardiol 1998; 82:26M31M] and the desirability of long-term permanent treatment [Terry A. Jacobson, Clinical Context: Current Concepts of Coronary Heart Disease Management, Am J Med. 2001; 110 (6A):3S11S], it becomes clear the necessity for search of new agents of similar targeting but with higher potency, safety and availability.
A promising area for search of the desirable agents includes naturally occurring sterol-like compounds and, especially, oxygenated sterols. One of the new possibilities of this type is presented by brassinosteroids, recently discovered class of plant hormones of steroid origin responsible for a wide spectrum of growth and adaptive reactions in plants [V. Khripach, V. Zhabinskii, A. de Groot, Brassinosteroids, Academic Press, San Diego, 1999]. As oxygenated derivatives of sterols structurally very close to cholesterol and its putative metabolites, brassinosteroids theoretically could be expected to fulfill both mentioned above functions on the prevention of high level cholesterol in blood: lowering absorption of exogenic cholesterol, on the one hand, and deactivation of the biosynthesis of the endogenic one, on the other hand. As ubiquitous plant constituents characteristic for all plant species, brassinosteroids were, and are, consumed by mammals with food throughout their evolution and, probably, play in them some regulatory role that is not recognized to date. This circumstance together with the data on low toxicity and absence of any direct and distant negative effects in animals indicate a possibility of their easy application to a solution of the task of the present invention without negative consequences for health of patients. Although being known more than twenty years, brassinosteroids have not yet been investigated as possible bio-regulators in humans, and this invention presents, to the best of our knowledge, the first attempt of such investigation and application of a brassinosteroid for human treatment, particularly, for lowering cholesterol level in blood and preventing in this way the risk of coronary heart disease.